Materials and method for inhibiting replication protein A and uses thereof

ABSTRACT

Targeting uncontrolled cell proliferation and resistance to DNA damaging chemotherapeutics with at least one reagent has significant potential in cancer treatment. Replication Protein A, the eukaryotic single-strand (ss) DNA binding protein, is essential for genomic maintenance and stability via roles in both DNA replication and repair. Reported herein are small molecules that inhibits the in vitro, in vivo, and cellular ssDNA binding activity of RPA, thereby disrupting the eukaryotic cell cycle, inducing cytotoxicity and increasing the efficacy of chemotherapeutic agents damage DNA, and/or disrupt its replication and/or function. These results provide new insights into the mechanism of RPA-ssDNA interactions in chromosome maintenance and stability. This represents a molecularly targeted eukaryotic DNA binding inhibitor and demonstrates the utility of targeting a protein-DNA interaction as a means of studying the cell cycle and providing a therapeutic strategy for cancer treatment.

PRIORITY CLAIM

This application is a divisional of U.S. patent application Ser. No.13/576,023, filed Oct. 15, 2012, which is a §371 national stageapplication of International Application Number PCT/US2011/023838, filedFeb. 5, 2011, titled Materials and Methods for Inhibiting ReplicationProtein A and Uses Thereof, which claims the benefit of U.S. ProvisionalPat. App. No. 61/301,778 filed on Feb. 5, 2010, and the disclosure ofeach application is expressly incorporated herein by reference in itsentirety.

STATEMENT OF GOVERNMENTAL RIGHTS

This invention was made with government support under CA082741 awardedby the National Institutes of Health. The Government has certain rightsin the invention.

FIELD OF THE INVENTION

Some aspects of the invention relate to identifying molecules that atleast partially inhibit the activity of the Replication Protein A andthese molecules can be used to treat hyper-proliferative diseases,including cancer.

BACKGROUND

Replication protein A (RPA) is a heterotrimeric single-stranded DNA(ssDNA) binding protein made up of 70, 34, and 14 kDa subunits (1). ThessDNA binding activity of RPA is required for several DNA metabolicpathways including DNA replication, recombination and repair. Highaffinity interactions with DNA are sustained by the numerousoligosaccharide/oligonucleotide binding (OB)-folds present on each ofthe three subunits (2; 3). The DNA binding pocket of a single OB-foldaccommodates 3-4 bases of ssDNA (4; 5). The main OB-folds, DNA bindingdomains A and B (DBD-A and DBD-B) are present in the central region ofthe p70 subunit and contribute most of the binding energy for RPA-ssDNAinteractions (2). Individual OB-folds are compact modular domainspopulated with hydrophobic and basic amino acids. These structuralfeatures make the OB-folds an attractive target for development of smallmolecule inhibitors (SMIs) of DNA binding activity. Given RPA's centralrole in cell growth and DNA repair, it is an attractive target for thedevelopment of compounds that can interfere with its activity. Someaspects of the instant invention include compounds that interact withRPA and methods of using the same to influence cell growth and death.

SUMMARY

Various aspects of the invention include methods and compounds forreducing the activity of Replication Protein A, effecting eukaryoticcell proliferation, the cell cycle of eukaryotic cells and/or treatingcancer by contacting Replication Protein A with a compound such ascompound A or a metabolite thereof that includes the following corestructure:

In some embodiments eukaryotic cells contacted with a compound ormetabolite thereof includes the core structure are further contactedwith at least one compound that damage DNA directly or inhibitstopoisomerase II. Such compounds include, but are not limited to,Cisplatin, Etoposide, Busulfan, Bendamustine, Carboplatin, Carmustine,Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Daunorubicin,Decitabine, Doxorubicin, Epirubicin, Etoposide, Idarubicin, Ifosfamide,Irinotecan, Lomustine, Mechlorethamine, Melphalan, Mitomycin C,Mitoxantrone, Oxaliplatin, Temozolomide, Topotecan and the like.

Some aspects of the invention include methods of reducing the activityof a Replication Protein A, comprising the steps of: providing acompound A, wherein the compound A binds to Replication Protein A or ismetabolized into a chemical that binds to Replication Protein A,compound have the following formula:

wherein, R₁ is selected from the group consisting of: substitutedquinolins, thiophenes and phenyls; including5-(7-chloro-2,3-dihydro-[1,4]dioxino[2,3-g]quinolin-8-yl);5-(quinoxalin-6-yl); and 6-chloro-[1,3]dioxolo[4,5-g]quinolin-7-yl R₂ isselected from the group consisting of: hydrogen, halogens, methylgroups, nitro groups; wherein compound A or a metabolite of compound Abinds to Replication Protein A; and R₃ is selected from the groupconsisting of: ketobutyric acids, and

wherein n=1, 2, 3, 4, or 5. In some embodiments the quinolin or thephenyl in compound A is substituted with at least one moiety selectedfrom the group consisting of: halogens, methyl groups, ethyl groups,amino groups and pyrazole. In some embodiments R₁ in compound A isselected from the group consisting of: 2-chloro-7-ethoxyquinolin,5(quinoxalin-6-yl), 2-chloro-6,7-dimethoxyquinolin,2-chloro-6-ethoxyquinolin, 4-Bromophenyl, 4-fluorophenyl,4-chlorophenyl, 2-chlorophenyl, 2-methoxyphenyl, 4-ethylphenyl,4-Diethylaminophenyl, 4-dimethylaminophenyl, Thiophene, 4-methoxyphenyl,p-tolyl, 2,5-dimethoxyphenyl, 3,4-dimethoxyphenyl, 4-ethylphenyl; and1-phenyl-3-p-tolyl-1H-pyrazole; and R₂ in compound A is selected fromthe groups consisting of: 4-bromo, 4-chloro, 4-nitro, 4-methyl,4-methoxy, H, 3,4-dimethyl.

In some embodiments the molecule that binds to Replication Protein A isa derivative of compound A or a metabolite thereof. And in someembodiments the molecule that binds to Replication Protein A is thecompound TDLR-505, the compound TDLR-506, or a metabolite thereof.

In still other embodiments the step of contacting either compound A or ametabolite thereof and the at least one isoform of Replication Protein Aoccurs in vivo. In still other embodiments the step of contactingcompound A or a metabolite thereof and the at least one isoform ofReplication Protein A occurs in vitro.

Other aspects of the invention include methods of inhibiting cellproliferation via altering the eukaryotic cell cycle-progression,comprising the steps of: providing a compound A that interferes witheukaryotic cell cycle-progression or that is metabolized into a chemicalthat interferes with eukaryotic cell cycle-progression, wherein compoundA has the following formula:

wherein, R₁ is selected from the group consisting of: substitutedquinolins, thiophenes and phenyls; including5-(7-chloro-2,3-dihydro-[1,4]dioxino[2,3-g]quinolin-8-yl);5-(quinoxalin-6-yl); and 6-chloro-[1,3]dioxolo[4,5-g]quinolin-7-yl; R₂is selected from the group consisting of: hydrogen, halogens, methylgroups, nitro groups; wherein compound A or a metabolite of compound Abinds to Replication Protein A; and R₃ is selected from the groupconsisting of: ketobutyric acid, and

wherein n=1, 2, 3, 4, or 5. In some embodiments the quinolin or thephenyl in compound A is substituted with at least one moiety selectedfrom the group consisting of: halogens, methyl groups, ethyl groups,amino groups and pyrazole. In some embodiments R₁ in compound A isselected from the group consisting of: 2-chloro-7-ethoxyquinolin,5(quinoxalin-6-yl), 2-chloro-6,7-dimethoxyquinolin,2-chloro-6-ethoxyquinolin, 4-Bromophenyl, 4-fluorophenyl,4-chlorophenyl, 2-chlorophenyl, 2-methoxyphenyl, 4-ethylphenyl,4-Diethylaminophenyl, 4-dimethylaminophenyl, Thiophene, 4-methoxyphenyl,p-tolyl, 2,5-dimethoxyphenyl, 3,4-dimethoxyphenyl, 4-ethylphenyl; and1-phenyl-3-p-tolyl-1H-pyrazole; and R₂ in compound A is selected fromthe groups consisting of: 4-bromo, 4-chloro, 4-nitro, 4-methyl,4-methoxy, H, 3,4-dimethyl.

In some embodiments the compound that at least partially interferes withthe eukaryotic cell cycle-progression is the compound A or a metabolitethereof. In still other embodiments the compound that interferes witheukaryotic cell cycle-progression is the compound TDLR-505, TDLR-506, ormetabolite thereof. In some embodiments, the contacting step betweencompound A or a metabolite thereof and the Replication Protein A occursin vivo.

Still other embodiments include methods for treating human or animalpatients comprising the step of supplying or providing at least onetherapeutically active amount of at least one compound that inhibits theactivity of RPA or a pharmaceutically acceptable salt thereof. Someembodiments include the step of administering a dose of the compound toa patient wherein the dose is about 50 mg of said compound per kg⁻¹ ofthe patient's body weight or about 100 mg of said compound per kg⁻¹ ofthe patient's body weight or about 200 mg of said compound per kg⁻¹ ofthe patient's body weight.

In still other embodiments the contacting step between said compound Aor a metabolite thereof and the Replication Protein A occurs in vitro.Still other aspects of the invention include a method treating cancer,comprising the steps of: providing a compound wherein the compoundinterferes with the cell cycle of a cancer cell or is metabolized into achemical that interferes with the cell cycle of a cancer cell, and thecompound has the following formula:

in which R₁ is selected from the group consisting of: substitutedquinolins, thiophenes and phenyls, and R₂ in compound A is selected fromthe group consisting of: halogens, methyl groups, nitro groups; whereincompound A binds to Replication Protein A; and contacting said compoundA with at least one cancer cell. In some embodiment the quinolin or thephenyl in compound A is substituted with at least one moiety selectedfrom the group consisting of: halogens, methyl groups, ethyl groups,amino groups and pyrazole. In some embodiments R₁ in compound A isselected from the group consisting of: 2-chloro-7-ethoxyquinolin,5(quinoxalin-6-yl), 2-chloro-6,7-dimethoxyquinolin,2-chloro-6-ethoxyquinolin, 4-Bromophenyl, 4-fluorophenyl,4-chlorophenyl, 2-chlorophenyl, 2-methoxyphenyl, 4-ethylphenyl,4-Diethylaminophenyl, 4-dimethylaminophenyl, Thiophene, 4-methoxyphenyl,p-tolyl, 2,5-dimethoxyphenyl, 3,4-dimethoxyphenyl, 4-ethylphenyl; and1-phenyl-3-p-tolyl-1H-pyrazole; and R₂ in compound A is selected fromthe groups consisting of: 4-bromo, 4-chloro, 4-nitro, 4-methyl,4-methoxy, H, 3,4-dimethyl. In some embodiments the compound that iscontacted with the cancer cell is compound A or a metabolite thereof.And in some embodiments the compound that is contacted with the cancercell is the compound TDLR-505, TDLR-506, or metabolite thereof. In someembodiments the contacting step between said compound or a metabolitethereof and cancer cell occurs in vivo. And in still other embodimentsthe contacting step between said compound A or a metabolite thereof andthe cancer cell occurs in vitro. In some embodiments the cancer cell isfound in a solid tumor selected from the group consisting of: lungcancer, non-small cell, small cell, epithelial ovarian cancer, cervicalcancer, colon cancer and breast cancer. Some embodiments of theinvention further include the step of contacting the cancer cell with atleast one chemotherapeutic reagent that binds to or damages DNA directlyor reduces the activity of topoisomerase II. In some embodiments thechemotherapeutic reagent is selected from the group consisting of butnot limited to: cisplatin and etoposide. Such compounds include, but arenot limited to, Busulfan, Bendamustine, Carboplatin, Carmustine,Cisplatin, Cyclophosphamide, Dacarbazine, Daunorubicin, Decitabine,Doxorubicin, Epirubicin, Etoposide, Idarubicin, Ifosfamide, Irinotecan,Lomustine, Mechlorethamine, Melphalan, Mitomycin C, Mitoxantrone,Oxaliplatin, Temozolomide, Topotecan and the like.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Compound 3.

FIG. 1B. EMSA of compound 3 inhibition of RPA binding to DNA.

FIG. 1C. Plot of EMSA data of compound 3 inhibition of RPA binding toDNA.

FIG. 2A. A ketobutyric acid derivative of compound A.

FIG. 2B. Compound TDLR-505.

FIG. 2C. EMSA of compound TDLR-505 inhibition of RPA binding to DNA.

FIG. 2D. Plot of EMSA data of TDRL-505 inhibition of RPA binding to DNA.

FIG. 3. EMSA of compound TDLR-506 inhibition of RPA binding to DNA.

FIG. 4A. Dot plots of H460 cells stained with Annexin V/PI measuringcell death induced by TDRL-505.

FIG. 4B. Plot of cell death data measured at different concentrations ofcompound TDLR-505.

FIG. 4C. Immuno-fluorescence of RPA cellular localization probed with ananti-RPA antibody and visualized with an AlexaFlour antibody.

FIG. 4D. Western blots of the of the p34 subunit of RPA as a function oftreatment with compound TDRL-505.

FIG. 5A. Cell cycle distribution of H460 NSCLC cells treated withcompound TDLR-505.

FIG. 5B. Cell cycle distribution of H460 cells treated with Nocodazoleand then compound TDLR-505 for 4, 8 or 12 hours.

FIG. 6. Analysis of synergy in H460 cells co-treated with compoundTDLR-505 and either cisplatin (open circles) or etoposide (closedcircles).

FIG. 7A. In vivo anticancer activity of TDRL-505 versus non-small celllung cancer in a mouse xenograft model. Tumor volume was measured andplotted versus time. Data are presented as the mean±S.E (n=5).

FIG. 7B. In vivo anticancer activity of TDRL-505 versus non-small celllung cancer in an individual mouse assessed in the xenograft model.

DESCRIPTION

For the purposes of promoting an understanding of the principles of thenovel technology, reference will now be made to the preferredembodiments thereof, and specific language will be used to describe thesame. It will nevertheless be understood that no limitation of the scopeof the novel technology is thereby intended, such alterations,modifications, and further applications of the principles of the noveltechnology being contemplated as would normally occur to one skilled inthe art to which the novel technology relates.

Unless specifically or implicitly stated otherwise the term ‘about’ asused herein means plus or minus 10 percent. For example, ‘about 1.0’encompasses the range of 0.9 to 1.1.

A therapeutically effective amount is an amount of a biologically activecompound that has a single or cumulative beneficial effect on the healthor well being of a patient.

Inhibiting RPA-DNA interactions has the potential to impact numerousdifferentially regulated pathways in cancer cells. In DNA replication,RPA inhibition can be used to exploit the highly proliferative nature ofcancer cells which is characterized by a large population of cells inS-phase. RPA is also essential for several DNA repair pathways in thecell including nucleotide excision repair (NER). Cisplatin, a commonchemotherapeutic used in the treatment of various cancers, induces itscytotoxic effect by forming intrastrand covalent DNA adducts that arerepaired primarily by the NER pathway (6). Consequently, cisplatintreatment, in conjunction with decreased RPA ssDNA binding activity,would be expected to result in decreased efficiency of cellular repairof cisplatin-DNA adducts and increased cytotoxicity. Thus, targeting RPAhas the potential not only for single agent activity but also tosensitize cancer cells to therapies that induce DNA damage and geneticinstability, such as cisplatin, etoposide and ionizing radiation (IR).Busulfan, Bendamustine, Carboplatin, Carmustine, Cisplatin,Cyclophosphamide, Dacarbazine, Daunorubicin, Decitabine, Doxorubicin,Epirubicin, Etoposide, Idarubicin, Ifosfamide, Irinotecan, Lomustine,Mechlorethamine, Melphalan, Mitomycin C, Mitoxantrone, Oxaliplatin,Temozolomide, Topotecan and the like.

As disclosed herein, small molecules that inhibits the ssDNA bindingactivity of RPA have been identified. Cellular RPA inhibition results inthe inability to enter S phase, induction of cell death and synergisticactivity with the chemotherapeutic reagents cisplatin and etoposide.These small molecules which are able to inhibit the ssDNA bindingactivity of RPA are active both as single agents and in conjunction withcommonly used chemotherapeutics for killing cancer cells. In vivo, thecompounds can be safely administered up to 200 mg/mg in mice IP and viaoral gavage with not signs of overt toxicity and possess anticanceractivity versus human non-small cell lung cancer in mouse xenograftmodel.

Materials and Methods

Synthesis of TDRL 505 Derivatives

Referring now to Scheme 1. Commercially available ketones such as 1 andaldehydes such as 2 care subjected to a Claisen-Schmidt condensation tocreate en-ones such as 3 that can be cyclized using reagents such ashydrazine to the generate H1 pyrazoles such as 4. Amide bond chemistryis used with various acids such as 5 in order to modify the N1 positionof the pyrazole to form compounds such as the exemplary compoundTDRL-505 6.

Synthesis of TDRL 506 Derivatives

Referring now to Scheme 2. TDRL-506 was prepared from TDRL-505 and3-morpholinopropan-1-amine (2) via EDC coupling.

In Vitro Analysis

Small molecule inhibitors were obtained from ChemDiv and resuspended inDMSO. Compound TDLR-505 was independently synthesized and structureconfirmed by mass spectrometry analysis. Human RPA was purified using anE. coli over expression system as previously described (7). EMSAs wereperformed in 20 μL reactions containing 25 nM RPA, 25 nM 5′[³²P]-labeled34-base pair DNA as previously described (7). The final concentration ofDMSO was 1%.

Flow Cytometry

H460 cells were analyzed for apoptosis using an Annexin V-FITC/Propidiumiodide (PI) Vybrant Apoptosis Assay Kit (Invitrogen), according tomanufacturer's instructions. Cells were plated at a density of 1×10⁴cells per cm² and allowed to adhere for 24 hours and then treated withcompound TDLR-505 for 48 hours. Following plating and treatment of H460cells as described above, adherent and non-adherent cells werecollected, processed, and analyzed on a BD FACScan flow cytometer. Datawas analyzed using WinMDI software(http://facs.scripps.edu/software.html). Cell cycle analysis wasperformed by PI staining Briefly, cells were plated and treated withcompound collected and then washed twice with PBS-EDTA supplemented with1% BSA. Cells were fixed in 70% EtOH at −20° C. while vortexing followedby incubation on ice for 30 minutes. Cells were then collected andstained with PI solution (10 μg/mL PI and 25 μg/mL RNaseA in PBS-EDTAsupplemented with 1% BSA). Cells were analyzed on a Becton DickinsonFACScan flow cytometer. Cells were gated and analyzed on a histogramwith events plotted against the FL2-A parameter. Cell cycle distributionwas analyzed using ModFit software. G2 arrest was induced by treatmentwith 0.8 μg/mL nocodazole for 12 hours (10). Cells were then washed withPBS and treated with either vehicle or compound TDLR-505 (100 μM). Cellswere harvested and analyzed for cell cycle distribution as describedabove.

Indirect Immunofluorescence

H460 cells were plated on chamber slides (LabTek) as described above.Cells were then treated for 3 hours with either 50 μM of compoundTDLR-505 or vehicle as indicated and following treatment, cells werefixed in 4% paraformaldehyde at 25° C. for 3 minutes followed by washingin 0.2% Triton X-100 for 2 minutes at 4° C. The slides were then blockedin 15% FBS in PBS for 1 hour at 25° C. and then incubated with anti-RPA34 primary antibody (Neomarkers) at a dilution of 1:500 in 15% FBS for 1hour. Slides were then washed 3×10 minutes with 15% FBS and thenincubated with Alexa Fluor-594 goat-anti-mouse secondary antibody(Invitrogen) at a dilution of 1:300 for 1 hour. Slides were again washedand stained with 300 nM DAPI diluted in PBS-EDTA for five minutes.Slides were then mounted and images captured using a Zeiss fluorescentmicroscope and images were captured using filters for Texas Red tovisualize RPA staining and DAPI for visualizing DNA. Slides werevisualized and images analyzed and quantified using ImageJ software.

Western Blot Analysis

H460 cells were plated and treated with either vehicle or 100 μMcompound TDLR-505 in the presence or absence of 25 μM etoposide for 6hours and then processed for western blot analysis using a RIPA lysisand extraction procedure. RPA was detected with an anti-RPA p34 antibody(Neomarkers) and goat anti-mouse-HRP secondary (Santa Cruz). Bands werevisualized using chemiluminescence detection.

In Vivo Analysis

NOD/SCID mice (8 weeks of age) were administered IP injections ofcompound TDLR-505 2× per week for 2 weeks at 200 mg/kg. Mice weremonitored for signs of overt toxicity and bodyweights measured threetimes per week. Mice were sacrificed and gross necroscopy performed toassess organ weights and inspected for signs of toxicity. There was noindication that the TDLR-505 was toxic when it was orally administeredto mice at dosing level of about 200 mg/mg. NOD/SCID mice were thenimplanted with 5×10⁶ H460 cells in 0.2 ml of 50% Matrigelsubcutaneously. Tumors were allowed to develop for 7 days and TDRL-505was administered via oral gavage 2× per week for one week. Tumor volumeswere calculated by measuring the width and the length of the tumors andusing these measurements in the equation: vol.=1×w²/2.

Pharmacokinetic analysis. TDRL-505 was administered to mice at 200 mg/kgeither ip or po and blood drawn at 1, 2, 4, 8, 24 and 48 hours posttreatment. Serum concentration of TDRL-505 was measured using a HPLCMS/MS protocol and PK parameters analysis using on-compartmentalanalysis.

Results

Identification of a Small Molecule Inhibitor of RPA

Previous work led to the identification of a series of small moleculesfrom the NCI library that inhibited the DNA binding activity of RPA butshowed no cellular activity (8). A further study undertook screening ofa ChemDiv library using a fluorescence polarization (FP) modification tothe original assay (11). Referring now to FIGS. 1A, B, and C, compound 3was identified from the high-throughput screen and analyzed in asecondary assay using electrophoretic mobility shift assays (EMSAs) toconfirm inhibition. As illustrated by FIGS. 1B and 1C, significantinhibition of RPA DNA binding was observed via EMSA analysis andquantification of the data bears out this inhibition.

Referring now to FIGS. 2A and B. In view of the high level of in vitroinhibition of RPA observed using compounds in the core structure ofcompound A, the core structure was substituted with dihydropyrazole witha 4-oxo-butanoic acid at the N1 position and a phenyl substituent at theC3 to initiate analysis of structure activity relationships (SAR) andsearch for other compounds with cellular activity. Eighty-one analogswere identified and obtained from the ChemDiv library with differingsubstitutions off the phenyl ring (R₂) and varying substituents atposition C5 on the dihydropyrazole ring (R₁) (data not shown). Among thecompounds analyzed, compound TDRL-505 (FIG. 2B) was the most potent RPAinhibitor tested having an IC₅₀ value of 13 μM (TABLE1). A number ofcompounds, including TDRL 518 and TDRL 520-523 were also identified inthe secondary screen as having in vitro RPA inhibitory activity, or ashaving chemical properties similar to the small molecules that werefound to inhibit RPA activity. Still referring to TABLE 1, each compoundin Table 1 was titrated against RPA in vivo or in cell, in vitro todetermine its IC₅₀ values. As illustrated in Table 1, these compoundsshowed varying capacities for inhibiting RPA-ssDNA interactions (TABLE1). In order to determine cellular activity of each of the compounds,the induction of cell death was measured in a H460 NSCLC cell line andIC₅₀ values for each compound following a 48 hour exposure weredetermined. These data are also presented in TABLE 1 and reveal acorrelation between in vitro and cellular activity, consistent withcellular inhibition of RPA and indicating specificity for RPAinhibition. However, compound 523, which showed minimal inhibition ofRPA-ssDNA interactions in vitro, also displayed modest cellularactivity. This may be the result of the cell metabolizing the compoundto generate a more effective RPA inhibitor. Analysis of the cellular andin vitro inhibitory activity of the compound 3 analogs, showed thatcompound TDLR-505 displayed the lowest in vitro IC₅₀ value and was themost potent compound of those examined in cells. Accordingly, thecompound was selected for further investigation, including studying itsmechanism of action and the cellular effects that result from inhibitionof RPA-ssDNA interactions using a comprehensive series of in vitro, cellbased and in vivo assays.

Referring now to TABLE 1, the in vitro IC₅₀ was determined by EMSAanalysis as described in FIG. 1A. The cellular IC₅₀ value was determinedby treating H460 cells with the indicated compounds and analyzingannexinV/PI staining as described in Methods. The in vitro and cellulardata was analyzed using standard 4 parameter logistic-curve. The IC₅₀values and standard error of the fit were determined from this analysis.

Referring now to Table 2. The compounds in table 2 are commerciallyavailable and have also been demonstrated to inhibit RPA-DNA bindingactivity in vitro. Briefly, “m” has oxo-pentanoic acid modification anda dioxinol substitution off the quinoline; “n” has oxo-pentanoic acidmodification and quinoxalin-yl- replacing the quinoline; “r” has abutanoic acid and a dioxolo substitution off the quinoline.

Derivatives of compounds m and n, which include a butyric acid and themorpholiono derivatives are made. The p-methyl is replaced with ap-bromo on the phenyl ring. Derivatives of “r” with the Br-phenyl groupand the morpholino group are also made. The SAR data described hereinshows that making the butyric longer (e.g., pentanoic) appears to besomewhat detrimental to activity, but removal of the entire sidechainresults in a substantial loss of RPA inhibitory activity. Accordingly,shorter derivatives of the morpholino with fewer carbons spacers aredesirable.

In Vitro Inhibition of RPA's DNA Binding Activity Targeting DBD-A and Bin the 70 kDa Subunit of RPA.

Referring now to FIGS. 2C, D and E, EMSA analysis of compound TDLR-505,briefly, increasing concentrations of compound TDRL-505 werepre-incubated with RPA and DNA binding activity was assessed via EMSAusing a 34-base ssDNA substrate. Referring now to FIG. 2D,quantification of the gel presented in FIG. 2C. Referring now to FIG.2D, the average and standard deviation of each point are presented. Thedata was fit to a standard 4 parameter logistic curve with an N=4.

TABLE 1 Structure activity relationships of small molecule RPAinhibitors IC₅₀ (μM) Name Structure In vitro Cellular TDRL-505

12.9 ± 1.3 30.8 ± 1.7 TDRL-518

>100* NA TDRL-520

20.3 ± 10.7 49.9 ± 2.5 TDRL-521

71.7 ± 33.9 56.9 ± 6.7 TDRL-522

56.1 ± 6.7 38 ± 32*** TDRL-523

>100** 31.0 ± 5.2 *inhibition at the highest concentration tested (100μM) was 9%; **inhibition at the highest concentration tested (100 μM)was 36%; ***Maximum observed cytotoxicity was 80% of control

TABLE 2 Structure Properties

“m” 5-(5-(7-chloro-2,3-dihydro-[1,4]dioxino[2,3-g]quinolin-8-yl)-3-(p-tolyl)-4,5-dihydro-1H- pyrazol-1-yl)-5-oxopentanoic acid Chemical Formula: C₂₆H₂₄ClN₃O₅ Exact Mass:493.14 Molecular Weight: 493.94 m/z: 493.14 (100.0%), 495.14 (33.3%),494.14 (29.4%), 496.14 (9.2%), 495.15 (3.9%), 497.14 (1.6%) ElementalAnalysis: C, 63.22; H, 4.90; Cl, 7.18; N, 8.51; O, 16.20

“n” 5-oxo-5-(3-phenyl-5-(quinoxalin-6-yl)-4,5-dihydro-1H-pyrazol-1-yl)pentanoic acid Chemical Formula: C₂₂H₂₀N₄O₃Exact Mass: 388.15 Molecular Weight: 388.42 m/z: 388.15 (100.0%), 389.16(24.1%), 390.16 (3.4%), 389.15 (1.5%) Elemental Analysis: C, 68.03; H,5.19; N, 14.42; O, 12.36

“r” 4-(5-(6-chloro-[1,3]dioxolo[4,5-g]quinolin-7-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)- 4- oxobutanoic acidChemical Formula: C₂₄H₂₀ClN₃O₅ Exact Mass: 465.11 Molecular Weight:465.89 m/z: 465.11 (100.0%), 467.11 (33.3%), 466.11 (27.3%), 468.11(8.5%), 467.12 (3.3%), 469.11 (1.5%) Elemental Analysis: C, 61.87; H,4.33; Cl, 7.61; N, 9.02; O, 17.17Modification of TDRL-505; Analysis of TDRL-506

Referring now to Scheme 2. TDRL-506 was synthesized and purified andassessed for in vitro RPA inhibitory activity. Referring to FIG. 3,TDRL-506 was assessed for RPA inhibitory activity against both fulllength RPA heterotrimer Lanes 1-7 and the DBD-A/B construct (lanes8-12). The concentrations used were 25, 50, 75 and 100 M TDRL-506. Thedata demonstrate that the TDRL-506 compound containing the morpholinomodification retains full RPA inhibitory activity.

Induction Cancer Cell Death by TDRL-505.

Referring now to FIGS. 4A, 4B, 4C and 4D, the effects of compoundTDRL-505 on induction of cell death and cell cycle progression in a H460NSCLC cell line was measured. Briefly, H460 cells were treated withcompound TDRL-505 for 48 hours and analyzed for cell viability viastaining with AnnexinV/PI and presented as dot plots (see FIG. 3A).Referring now to FIG. 4B, the data from FIG. 4A was quantified and thepercentage of Annexin-/PI-cells (lower left quadrant), indicating livecells, was calculated. The average±SD (N=4) are presented and the datawas fit to a 4-parameter logistic curve. These data demonstrate thatTDRL-505 is capable of inducing cell death in NSCLC cells. As anindependent measure of the effect of compound TDLR-505 on cellviability, a crystal violet staining assay was used and gave an IC₅₀value of 64 μM (data not shown). A similar result was also observed intreatment of the A549 NSCLC cell line with compound TDLR-505 whileanalysis using freshly isolated peripheral blood mononuclear cells(PBMCs) revealed minimal cytotoxic activity (data not shown). In theseexperiments, compound TDLR-505 shows significant cytotoxic effects inNSCLC cell lines and only modest activity in non-cancerous cells,indicating that there is a therapeutic treatment window for thesemolecules.

These cellular effects seen following treatment with compound TDLR-505correlate with an inability of RPA to interact with DNA which could thenresult in numerous possibilities including degradation or redistributionof RPA within the cell. Indirect immunofluorescence was used to assesshow inhibition of RPA binding influences cellular localization.Referring now to FIG. 4C, H460 cells were treated with 50 μM compoundTDLR-505 or vehicle for 3 hours and analyzed for RPA expression andlocalization by indirect immunofluorescence using an Alexa Fluor594secondary antibody (red). Slides were counter stained with DAPI (blue)and images merged. Magnification of the boxed cells is presented belowthe low magnification images. Briefly, after 3 hours of treatment withcompound TDLR-505, cells showed a decrease in the intensity of RPAstaining compared to vehicle treated control without a change in overallsub-cellular localization. (FIG. 4C). Quantification of the intensity ofstaining revealed that of cells treated with vehicle, 23% showedintensity >70% of the maximum, while cells treated with compound showedonly 15% above that value. These data demonstrate that treatment ofcells with TDRL-505 reduces the amount of RPA bound to DNA in cells.Referring now to FIG. 4D, H460 cells were treated with vehicle or 100 μMcompound TDLR-505 without or with etoposide (25 μM) as indicated in thefigure. RPA expression was assessed via western blot analysis probingfor the p34 subunit. Lane 1 is a positive control with RPA purified froman E. coli expression system. The position of un-phosphorylated RPA p34and hyper-phosphorylated RPA p34 are indicated by the arrows. These datademonstrate that RPA is not degraded or reduced in expression as afunction of TDRL-505 treatment.

Compound TDLR-505 Induces a G1 Arrest in H460 Cell Lines.

The compound TDLR-505 was tested to assess its affect on cell cycleprogression. Knockdown of RPA by siRNA has been demonstrated to induce aG1 cell cycle arrest consistent with the essential role of RPA in theinitiation of S-phase DNA replication (19). In order to determine ifthis is the same mechanism of action displayed by compound TDLR-505, itsaffect on H460 cell cycle progression was measured. As illustrated bythe data presented in FIG. 5, synchronized H460 cells show an inabilityto re-enter S-phase when treated with TDRL-505.

Referring now to FIG. 5A. H460 NSCLC cells were treated with 0 and 75 μMof compound TDLR-505 for 48 hours and then analyzed for cell cycledistribution using flow cytometry. FIG. 5B, H460 cells were treated with0.8 μg/mL nocodazole for 12 hours, washed then treated with eithervehicle or 100 μMTDLR-505 for 4, 8 and 12 hours. Cells were thenharvested and analyzed for cell cycle distribution using flow cytometry.

The analysis of compound TDRL-505 on an asynchronous culture was testedand an increase in the proportion of cells in G1-phase was observed inresponse to treatment with TDRL-505 (FIG. 5A). To determine if entryinto S-phase is inhibited in compound TDLR-505 treated cells, cells weresynchronized in G2/M with nocodazole and then released from G2 arrestand re-fed complete medium supplemented with either vehicle or compoundTDLR-505. Referring now to FIG. 5B, both control and treated cellsrapidly progressed through mitosis into G1 after removal of nocodazole.Cells that were treated with vehicle alone entered into G1, as seen atthe 4 hour time point and progression into S-phase is apparent at the 8hour time point with progression into G2 evident at the 12 hour timepoint. Cells that were treated with compound TDLR-505 after release fromnocodazole progressed into G1 phase of the cell cycle but did not enterS-phase even 12 hours post release. These data demonstrate that TDRL-505induced a G1 cell cycle arrest in lung cancer cells.

Inhibition of RPA's Role in DNA Repair and Replication.

In addition to its essential role in DNA replication, RPA is requiredfor the repair of bulky DNA adducts as well as DNA breaks induced byvarious types of exogenous and endogenous agents. The association of RPAwith ssDNA is a critical feature of all of these pathways, indicatingthat inhibition of this activity would increase the cytotoxic effectsinduced by DNA damage. In order to determine the effect of RPAinhibition on cellular sensitivity to cisplatin, the combination index(CI) (20) was measured. Referring now to FIG. 6. As illustrated in thisexperiment, compound TDLR-505 acts synergistically with both cisplatinand etoposide in H460 cells. H460 cells were treated with increasingfractions of the IC₅₀ concentration of either cisplatin or etoposidewith compound TDLR-505 for 48 hours. After treatment, cells wereharvested and analyzed by annexin V/PI flow cytometry. Open circlesindicate CI analysis of cisplatin with TDLR-505 and closed circlesrepresent etoposide with TDLR-505. The combination index analysis wasperformed as previously described (20). The data are presented as theaverage±SD from (N=3). When cisplatin and compound TDLR-505 were used incombination, cell viability was decreased to a level that was greaterthan that induced by either agent alone, resulting in a synergy betweenthe two compounds and CI of 0.4 at the highest fraction of cellsaffected. The interaction became additive and then antagonistic(revealed from CI values greater than one) at lower fractions of cellsaffected. These results demonstrate that compound TDLR-505 is able topotentiate the effect of cisplatin in H460 cells and is consistent withinhibition of the cellular activity of RPA in NER. The ability ofcompound TDLR-505 to synergize with etoposide was also examined.Etoposide induces replication fork arrest and DNA damage response, bothcellular processes that require RPA (21). Using the same analysis asdescribed above for cisplatin, compound TDLR-505 showed synergisticactivity with etoposide at all fractions of cells affected (FIG. 6). RPAp34 has been shown to be hyperphosphorylated in response to etoposidetreatment in a variety of cellular systems (21; 22). Interestingly,analysis of RPA p34 hyperphosphorylation by western blot analysis wasnot altered by concurrent treatment with TDLR-505 and no dramatic changein overall RPA expression was evident (FIG. 4D). This data demonstratethat treatment with TDLR-505 does not dramatically impact etoposidedependent DNA damage signaling response and that synergy is observed inthe presence of RPA hyperphosphorylation.

In Vivo Analysis of Compounds TDLR-505.

Referring now to Table 3. TDRL-505 demonstrating reasonable IC₅₀ valuesagainst NSCLC prompted the assessment in vivo of the effect of RPAinhibition on lung cancer tumor growth. The safety of administration ofTDRL-505 in naive mice was determined Additional data demonstrated thatIP administration of compound TDLR-505 in a formulation of 25% DMSO in0.75% Tween 80 to NOD/SCID mice is well tolerated up to 400 mg/kg. Oraldosing in 0.1% Tween 80/methylcellulose was also well tolerated up to800 mg/kg.

Mice that had been implanted with a human NSCLC tumor cells weresubjected to a regime of oral dosing. The tumors were allowed to grow to100 mm3 after which treatment with TDRL-505 or vehicle was commenced.The results presented in (FIG. 7) demonstrate a reduction in tumorgrowth in mice treated with TDRL-505 compared to the vehicle control.Referring to FIG. 7B, analysis of individual mice reveal significantreduction in tumor growth compared to vehicle treated controls.

Based on the in vivo anticancer activity of TRL-505 a comparison ofpharmacokinetic parameters with both ip and po dosing regimens was made.Naive mice were given a single dose and as described in Table 3, bloodwas drawn at various time intervals and TRDL-505 concentrationdetermined via a HPCL/MSMS protocol developed for this compound. Thedata demonstrate significantly better PK parameters in nearly allparameters for ip dosing compared to po. These data suggest the ipdosing will yield significantly greater bioavailability and lead toenhanced efficacy leading to even greater anti cancer activity. Inaddition, the morpholino-modification employed in TDRL-506 shouldincrease is oral bioavailability thereby rendering more effective oraldosing regimens for TDRL-506.

TABLE 3 TDRL-505 Pharmacokinetics: Noncompartmental analysis Dose Weight(avg) C_(max) t_(max) AUC_(0-t) t_(1/2) Cl/F Vd_(ss) (mg/Kg) RouteFormulation (Kg) (ng/mL) (hours) (ng * mL⁻¹ * hr) (hours) (mL/hr) (L)200 IP Tween80/DMSO 0.0242 31702 8 577143 7.13 8.29 0.11 200 POMethylcellulose/ 0.0253 6397 1 24971 2.62 202.33 0.88 DMSO C_(max):maximum concentration t_(max): time of maximum concentration AUC_(0-t):area under the plasma concentration-time curve up to time, t Cl/F:clearance/availability, If dosage is IV, then F = 1. Vd_(ss): apparentvolume of distribution at steady state; Vd_(ss) = t_(1/2): half-life

Advances in high throughput screening and chemical libraries haveresulted in an explosion of putative cancer targets and theirinhibitors. To date, the majority of these target enzymatic activityassociated with a specific protein. As illustrated herein, targeting thenon-enzymatic DNA binding activity of RPA opens up an entire new classof putative interactions. Compound TDLR-505 blocks cell entry intoS-phase and results in a cytotoxic/cytostatic response, the result isconsistent with inhibiting RPA's role in the initiation of DNAreplication and this process likely involves a complex series ofinteractions, one of which is the loading of RPA at replication originsin a S-phase CDK dependent process (19; 23). While these datademonstrate no appreciable DNA synthesis, it remains to be determined ifRPA is able to load at origins in cells treated with compound TDLR-505.The exit of cells from S-phase into G2 in the presence of TDLR-505suggests that DBD-A and -B DNA interactions may be less important inelongation, in which RPA predominantly participates in lagging strandDNA synthesis (24). RPA's role in the early stages of DNA replicationwould predict a G1-arrest as opposed to an intra S-phase arrest, whichwe observed. However, the potential that S-phase cells treated withcompound TDLR-505 have reduced RPA binding and hence blocked elongationand firing of late replication forks cannot be ruled out. This presentsthe possibility that cells killed by compound TDLR-505 are thoseactively replicating their DNA and the interruption of this causes theobserved cytotoxicity. However, the continuation of G1 arrest may alsoinduce cell death after prolonged periods of time, explaining why thedegree of cell death after 48 hours of treatment is greater than thenumber of cells that are in S-phase. This illustrates the existence of atherapeutic window for specifically targeting actively dividing cells inthe context of cancer treatment using SMIs to block the cellularactivity of RPA.

The role of RPA in DNA repair also allows for inhibition of its activityto increase the efficacy of current chemotherapeutics that induce DNAdamage in the context of combination therapy. The inhibition of DNArepair is anticipated to result in persistent DNA damage which wouldincrease cytotoxicity. The indispensable role of RPA in the recognitionand verification steps of NER is well characterized and, in addition,RPA participates in the re-synthesis step following excision of thedamaged oligonucleotide (25). Previous studies have shown that cellswith decreased levels of NER proteins demonstrate increased sensitivityto cisplatin treatment (26). Consistent with this, our data reveal asynergistic interaction between compound TDLR-505 and cisplatin at highfractions of cells affected. Interestingly, at low fractions of cellsaffected, an antagonistic interaction is observed with combinationindices greater than one. This is likely the result of interactions notat the level of repair but at the level of signaling. As cisplatin leadsto activation of a G2 checkpoint and induces apoptosis from an extendedG2 arrest, the finding that compound TDLR-505 blocks cells in G1indicates that fewer cells would be subject to cisplatin induced G2arrest. Likewise, if compound TDLR-505 toxicity stems from an extendedG1 arrest, the G2 checkpoint induced by cisplatin would result in lesscell death as a result of treatment. At high concentrations, this effectis mitigated by the interaction at the level of DNA repair with RPAinhibition increasing cisplatin toxicity and overcoming the antagonisticsignaling interaction.

The role of RPA in DNA replication restart and processing of collapsedreplication forks also presents opportunities for combination therapy(27; 28). Interestingly, combination index analysis of the activity ofetoposide with compound TDLR-505 showed synergistic activity at allfractions of cells affected. Etoposide inhibits the enzymatic activityof topoisomerase II (top II) resulting in persistent covalent-cleavagecomplexes on DNA which lead to replication fork arrest and both singleand double strand breaks (29). RPA has been demonstrated to respond toand repair these types of lesions and DNA intermediates and inhibitingthis activity would be expected to potentiate the effects seen byinhibiting top II, which is observed in our analyses (21). Secondly, dueto the asynchronous nature of these cells, at any given time a cellundergoing replication would be expected to be in various stages ofreplication firing. RPA is required in early replication firing, whiletop II has been shown to be required for later stage replication events(30). Therefore, inhibition of both stages of replication progressionwould be expected to show a greater effect than inhibiting either one ofthe steps individually, which would implicate a synergistic relationshipbetween compound TDLR-505 and etoposide. Inhibition of RPA activity andabrogation of pathway function has the potential for widespread utilityin cancer treatment. The role of RPA in several other repair pathwaysopens up other opportunities for combination therapy using RPAinhibitor. For example, combining molecularly targeted RPA inhibitionwith radiation therapy could lead to increased cytotoxicity in tumorcells via inhibition of DNA double strand break repair vianon-homologous DNA end joining or homologous recombination, both ofwhich have been shown to require RPA (31-33).

While targeting the enzymatic activity of proteins with small moleculesis well accepted, the results presented herein demonstrate thefeasibility and utility of targeting a non-enzymatic protein-DNAinteraction. These compounds are Small Molecule Inhibitor (SMIs) of RPAwhich display both in vitro and cellular activity. The approach oftargeting RPA for cancer chemotherapy has several unique advantagesincluding the lack of redundancy resulting from the absence of efficientback-up systems necessary to counteract the loss of RPA activityInhibition of RPA will have broad spectrum utility as the reliance onRPA for increased cell proliferation and repair of chemotherapeutic DNAdamaging agents is not unique to any single cancer. Targeting the DNAbinding activity of RPA with small, drug-like molecules as disclosedherein illustrates that this class of proteins can be targeted toproduce therapeutic compounds.

While the novel technology has been illustrated and described in detailin the figures and foregoing description, the same is to be consideredas illustrative and not restrictive in character, it being understoodthat only the preferred embodiments have been shown and described andthat all changes and modifications that come within the spirit of thenovel technology are desired to be protected. As well, while the noveltechnology was illustrated using specific examples, theoreticalarguments, accounts, and illustrations, these illustrations and theaccompanying discussion should by no means be interpreted as limitingthe technology. All patents, patent applications, and references totexts, scientific treatises, publications, and the like referenced inthis application are incorporated herein by reference in their entirety.

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We claim:
 1. A method of treating lung cancer in a subject in needthereof, comprising: a. administering a compound of the formula

or a pharmaceutically acceptable salt thereof, wherein the compound, ora metabolite thereof, binds to Replication Protein A.
 2. The methodaccording to claim 1, wherein, the compound that binds to ReplicationProtein A is of the formula

or a pharmaceutically acceptable salt thereof or a metabolite thereof.3. The method according to claim 1, wherein, the compound that binds toReplication Protein A is of the formula

or a pharmaceutically acceptable salt thereof or a metabolite thereof.4. The method according to claim 1, wherein the compound is administeredorally.
 5. The method according to claim 4, wherein the compound is in aformulation comprising methylcellulose.
 6. The method according to claim1, wherein the compound is administered intraperitoneally.
 7. The methodaccording to claim 6, wherein the compound is in a formulationcomprising Tween-80.
 8. A The method of claim 1 wherein the subject is ahuman patient.
 9. The method of claim 1, wherein the subject is ananimal.
 10. The method according to claim 8, further comprising b.administering to the human patient a therapeutically effective amount ofa chemotherapeutic DNA damaging agent or a topoisomerase II inhibitor.11. The method according to claim 10, wherein the chemotherapeutic DNAdamaging agent is selected from the group consisting of Cisplatin,Etoposide, Busulfan, Bendamustine, Carboplatin, Carmustine,Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Daunorubicin,Decitabine, Doxorubicin, Epirubicin, Etoposide, Idarubicin, Ifosfamide,Irinotecan, Lomustine, Mechlorethamine, Melphalan, Mitomycin C,Mitoxantrone, Oxaliplatin, Temozolomide, and Topotecan.
 12. The methodaccording to claim 8, wherein the compound is administered in a dose ofabout 50 mg of said compound per kg of the human patient's body weight.13. The method according to claim 8, wherein the compound isadministered in a dose of about 100 mg of said compound per kg of thehuman patient's body weight.
 14. The method according to claim 8,wherein the compound is administered in a dose of about 200 mg of saidcompound per kg of the human patient's body weight.